This proposal seeks to understand mlecular mechanisms of short-term regulation of NHE3, the brush border member of the mammalian Na+/H exchanger isoform 3, as occurs in the kidney and intestine with changes in dietary Na+ intake. Short term regulation of NHE3 is shown to follow changes in intracellular second messengers. In our previous work (Zizak M. et al., 1999. J. Biol. Chem. 274, 24753-24758) we demonstrated that in fibroblasts, in which cAMP did not have any effect, cAMP inhibition of NHE3 could be reconstituted when the regulatory factors, NHERF or E3KARP, were present. That study provided new insights into a mechanism of NBE3 regulation by suggesting an action of regulatory factors in a signaling complex that includes NHE3, regulatory factor, ezrin and cAMP-dependent protein kinases. In this complex, regulatory factors act as a scaffold to assemble a signaling complex. cGMP was shown to be involved in regulation of Na+ absorption in some cells while in other cells it does not have any effect. The preliminary demonstration that both E3KARP and cGKII expression are necessary to make NHE3 sensitive to modulation by cGMP in cells which did not previously display a cGMP-inducible NHE3 regulation indicates that E3KARP and cGKII are key mediators of cGMP provoked inhibition of NHE3 in PS120NBE3V cells where both proteins, cGKII and E3KARP are co-expressed The observation that cGMP failed to regulate Na+/H+ exchange in the cGMP kinase II non-infected cells demonstrates that physiological activation of cGMP does not regulate Na+/H+ exchange by cross activation of cAMP dependent protein kmase. This is the first time that direct regulation of NHE3 by cGMP or cGMP kinases has been shown. Therefore, the major goal of this study is to increase understanding of short-term regulation of NBE3 by assessing the role of regulatory protein E3KARP in cGMP/cGMP dependent protein kmase lI inhibition of NHE3 which is important for both physiologic and pathophysiologic regulation of NBE3 in kidney and intestine. The hypothesis to be tested in this application is that a PDZ domain containing protein, E3KARP is necessary for cGMP protein kinase II inhibition of NHE3. Studies are proposed to understand how E3KARP carries out this function, testing whether it acts as a scaffold to assemble a signaling complex that includes NHE3 and cGMP kinase II and to determine whether E3KARP is the first identified functionally important cGMP kdnase II Anchoring Protein (GKAP).